CPSC Staff Statement on University of Cincinnati Report

“Toxicity Review for Bis(2-ethylhexyl) Adipate (DEHA)”1

October 2018

The U.S. Consumer Product Safety Commission (CPSC) contracted with the University of Cincinnati to conduct toxicology assessments for six dialkyl o-phthalate (o-DAP) substitutes: acetyl tri-n-butyl citrate (ATBC); bis(2-ethylhexyl) adipate (DEHA); di-2-ethylhexyl terephthalate (DEHT); 1,2-cyclohexanedicarboxylic acid, dinonyl ester, branched and linear (DINX); trioctyltrimellitate (TOTM); and 2,2,4-trimethyl-1,3-pentanediol-diisobutyrate (TPIB). The reports will be used to inform staff’s assessment of products that may contain these compounds and is the first step in the risk assessment process.

CPSC staff assesses a product’s potential health effects to consumers under the Federal Hazardous Substances Act (FHSA). The FHSA is risk-based. To be considered a “hazardous substance” under the FHSA, a consumer product must satisfy a two-part definition. First, it must be “toxic” under the FHSA, or present one of the other hazards enumerated in the statute. Second, it must have the potential to cause “substantial personal injury or substantial illness during or as a proximate result of any customary or reasonably foreseeable handling or use.” Therefore, exposure and risk must be considered in addition to toxicity when assessing potential hazards of products under the FHSA.

The first step in the risk assessment process is hazard identification, which consists of a review of the available toxicity data for the chemical. If it is concluded that a substance may be “toxic”, then a quantitative assessment of exposure and risk is performed to evaluate whether a specified product may be considered a “hazardous substance”.

The toxicity review for DEHA follows.

1 This statement was prepared by the CPSC staff, and the attached report was produced by the University of Cincinnati for CPSC staff. The statement and report have not been reviewed or approved by, and do not necessarily represent the views of, the Commission.

TOXICITY REVIEW FOR

BIS(2-ETHYLHEXYL)ADIPATE

(DEHA)

Contract No. CPSC-D-17-0001

Task Order No. 003

Prepared by:

Risk Science Center

Department of Environmental Health

University of Cincinnati

160 Panzeca Way, Room G24

Cincinnati, OH 45267

Prepared for:

Kent R. Carlson, Ph.D.

U.S. Consumer Product Safety Commission

4330 East West Highway

Bethesda, MD 20814

August 8, 2018

* This report was prepared for the Commission pursuant to contract CPSC-D-17-0001 It has not been reviewed or approved by, and may not necessarily reflect the views of, the Commission.

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Table of Contents

1 Introduction ............................................................................................................................. 4

2 Physico-Chemical Characteristics ........................................................................................... 5

3 Manufacture, Supply, and Use ................................................................................................ 6

4 Toxicokinetics ......................................................................................................................... 8

5 Hazard Information ................................................................................................................ 10

5.1 Acute Single Dose Toxicity ........................................................................................... 10

5.1.1 Acute Oral Toxicity .................................................................................................... 10

5.1.2 Acute Dermal Toxicity ............................................................................................... 11

5.1.3 Acute Inhalation Toxicity ........................................................................................... 11

5.1.4 Irritation/Sensitization ................................................................................................ 11

5.2 Repeated Dose Toxicity ................................................................................................. 12

5.3 Chronic Toxicity/Carcinogenicity .................................................................................. 14

5.4 Reproductive Toxicity .................................................................................................... 17

5.5 Prenatal, Perinatal, and Postnatal Toxicity..................................................................... 20

5.6 Genotoxicity ................................................................................................................... 22

5.7 Mechanistic Studies........................................................................................................ 23

5.8 Mode of Action (MOA) ................................................................................................. 24

5.9 Lowest Hazard Endpoints by Organ System and Exposure Duration ........................... 25

5.10 Uncertainties and Data Gaps .......................................................................................... 26

6 Exposure ................................................................................................................................ 34

7 Discussion .............................................................................................................................. 36

7.1 Toxicity Under FHSA ........................................................................................................ 36

8 References ............................................................................................................................. 37

Appendix 1. Literature Search Terms Used …………………………………………..…………47

Appendix 2. Explanation of Physico-chemical Parameters .......................................................... 48

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1 Introduction

This report summarizes available data on the identity, physicochemical properties, manufacture,

supply, use, toxicity, and exposure associated with bis(2-ethylhexyl)adipate (DEHA). It is an

update of a previous contractor report to CPSC (Versar, 2010).

Literature searches for physico-chemical, toxicological, exposure, and risk information were

performed in November 2017 using the CAS number and synonyms (see Appendix 1 for the full

list of search terms), and using the following databases:

EPA SRS

PUBMED

RTECS

TSCATS (included in TOXLINE)

TOXNET databases, including

o TOXLINE

o CCRIS

o DART/ETIC

o GENE-TOX

o HSDB

Searches of the PubMed and Toxline databases covered all dates through the date of the search

(November, 2017). However, studies dated up to 2007 were screened out of the library during

the screening process using the Endnote files, as the current report supplements and updates a

staff report prepared in 2010 (Versar, 2010). Other databases and websites were also used to

identify additional key information, particularly authoritative reviews. Searches for authoritative

reviews addressing general toxicity and physicochemical information were conducted with the

following databases using the CAS number for DEHA and synonyms. These sites included:

ANSES Information on Chemicals (https://www.anses.fr/en)

ChemIDPlus (https://chem.nlm.nih.gov/chemidplus/)

ECHA Information on Chemicals (https://echa.europa.eu/information-on-chemicals)

EFSA (https://www.efsa.europa.eu/)

EPA (https://www.epa.gov/)

EPA chemistry dashboard (https://comptox.epa.gov/dashboard)

EPA IRIS (https://www.epa.gov/iris)

FDA (https://www.fda.gov/)

Google

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Health Canada (https://www.canada.ca/en/health-canada.html)

IARC (https://www.iarc.fr/)

INCHEM (http://www.inchem.org/)

JECFA (http://www.who.int/foodsafety/areas_work/chemical-risks/jecfa/en/)

NICNAS (https://www.nicnas.gov.au/)

NTP (https://ntp.niehs.nih.gov/)

OECD (http://www.oecd.org/)

WHO (http://www.who.int/en/)

Two new DEHA toxicology studies were identified in the literature searches. These were an

evaluation of ovarian toxicity, female fertility and developmental toxicity in rats (Wato et al.,

2009) and a developmental toxicity study in rabbits (Anonymous, 2014, as cited by ECHA,

2018). Other new studies that were found in the primary literature included studies on

toxicokinetics, exposure and mechanism of action, as well as reviews. Several of the key

toxicity studies were unpublished and not available as the primary studies. Therefore, these

studies were evaluated based on authoritative reviews and data compilations, including

SCENIHR (2008), Danish EPA (2010), OECD (2012), ANSES (2015), ECHA (2018), and

Eastman Chemical (2010).

Several additional review publications have been published since the previous CPSC assessment

(Versar, 2010). Reviews and posted data from ECHA (2018) provided useful new information.

2 Physico-Chemical Characteristics

DEHA is an ester of 2-ethylhexanol and adipic acid. Physical-chemical properties for this

compound are highlighted in Table 1.

Table 1: Physicochemical Properties and Identification Information for Di(2-ethylhexyl)

Adipate

Chemical Name Di(2-ethylhexyl) adipate

Synonyms Hexanedioic acid, 1,6-bis(2-ethylhexyl) ester; Bis(2-ethylhexyl)

hexanedioate; Di-(2-ethylhexyl) adipate; Dioctyl adipate;

Hexanedioic acid, bis(2-ethylhexyl) ester; Adipic acid, bis(2-

ethylhexyl) ester; Bis(2-ethylhexyl) adipate; DEHA; Di(2-ethylhexyl)

adipate; Di(2-ethylhexyl) adipate; Di-2-ethylhexyl adipate;

Hexanedioic acid, dioctyl ester; Octyl adipate

CAS Number 103-23-1

Structure

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(EPA Chemistry Dashboard)

Chemical Formula C22H42O4

Molecular Weight 370.574 g/mol

Physical State Liquid (MSDS Eastman Chemical, 2014)

Color Colorless (MSDS Eastman Chemical, 2014)

Melting Point -67.8°C

Boiling Point 417°C

Vapor Pressure 8.50E-07 mm Hg @ 20°C

Water Solubility <0.005 mg/L @ 22°C (OECD SIDS)

Log Kow 6.83

Flashpoint 175°C (median; EPA Chemistry Dashboard)

Source ChemIDplus (unless otherwise stated)

Kow is the octanol-water partition coefficient. See Appendix 2 for more detail.

DEHA is also known as dioctyl adipate (DOA), hexanedioic acid, and bis(2-ethylhexyl)

hexanedioate in some documents cited below.

The vapor pressure for DEHA indicates that in the atmosphere it may exist in both the gas and

particle phases. It will be removed from the air via dry and wet deposition or via degradation

primarily taking place through reactions with hydroxyl radicals. Direct photolysis is also a

possible degradation route, because of functional groups on the molecule that absorb UV-light

(HSDB, 2008).

The water solubility of DEHA, based on a slow stir and saturator column methods, is estimated

to be <0.005 mg/L (OECD SIDS). This estimate is considerably lower than the Ksol reported by

HSDB (0.78 mg/L), which was likely determined by the vigorous shaking method, which can

produce an emulsion rather than a solution. The lower Ksol estimate is more consistent with the

solubility of other structural analogs and the high log Kow (predicted value based on structure;

Table 1). DEHA has a relatively high Koc value, indicating that it will sorb to organic carbon

(Remberger et al., 2005). This, combined with its low vapor pressure, explains why DEHA is

considered to be immobile when released to soil (HSDB, 2008). In the water environment,

DEHA will sorb to particles and end up in the sediment, thus its transport via water is expected

to be limited (HSDB, 2008). However, DEHA, like all adipates, is able to undergo hydrolysis,

increasing its water solubility (HSDB, 2008). The BCF for DEHA is low, at 27 L/kg. In general,

adipates, including DEHA, are fairly reactive substances, which readily degrade both in the

environment and in organisms (Remberger et al., 2005).

3 Manufacture, Supply, and Use

Manufacture and Supply

DEHA is an EPA High Production Volume chemical, indicating an annual production volume or

importation volume above 1 million pounds in the U.S. (HPVIS, 2008).

Use

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DEHA is a commonly used plasticizer in lubricants, glue, scotch-tape, and sealants (Remberger

et al. 2005). In particular, it is used extensively as a plasticizer in flexible polyvinyl chloride

(PVC) and food contact films (Silva et al. 2013). It is also used in wire cable tubing, footwear,

vinyl flooring, stationery, wood veneers, coated fabrics, gloves, artificial leather, carpet backing,

and possibly toys (NICNAS, 2011; Bui et al., 2016). Unlike other adipates permitted for use as

acidity regulator food additives, the U.S. FDA regulation allows DEHA only as an indirect food

additive as a component of adhesives (FDA, 1999; HSDB, 2008).

As early as 2002, DEHA’s presence was detected in children’s soft PVC articles (Chen, 2002).

In that study, the Consumer Product Safety Commission’s Directorate for Laboratory Sciences

purchased 41 children’s products from retail stores, one of which was analytically identified as

containing DEHA (Chen, 2002). However, a more recent study (Dreyfus, 2010, as cited by

CPSC, 2014) did not find DEHA in any toys or childcare items. DEHA can also be found in a

variety of home and office products, such as vinyl flooring, carpet backing, wood veneer, and

coated fabrics (SCENIHR, 2008).

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4 Toxicokinetics

Absorption

DEHA is readily absorbed in mice, rats and monkeys (ECHA, 2018). B6C3F1 mice (4/sex/dose)

gavaged with a dose of 50, 500 or 5000 mg/kg 14C-labeled DEHA rapidly absorbed DEHA (or

its metabolites) from their GI tracts. At the low and mid doses, approximately 91% of the

administered dose was eliminated in urine within 24 hours. Approximately 7-8% of the

administered dose was eliminated in the feces. At the highest dose level (5000 mg/kg),

approximately 75% of the administered dose was eliminated in the urine, and 4% in the feces.

Although these studies indicate total oral absorption of radio-labeled 14C-DEHA is ≥ 90%, these

values do not indicate the systemic bioavailability of the parent DEHA itself by the oral route.

That is, the amount of radiolabel absorbed is not informative as to whether the radioactivity is in

the form of the parent or metabolite, and thus not informative as to the amount of parent in the

blood.

DEHA has a LogPO:W of approximately 9, predicting low percutaneous absorption. LogPO:W

describes the partitioning of a chemical between an aqueous phase (e.g., vehicle) and a lipid

phase (e.g., stratum corneum), assuming skin permeability is directly proportional this partition

coefficient. This prediction of low percutaneous absorption was supported by a study that

evaluated human bioavailability of DEHA in vitro under conditions mimicking occlusive skin

application. Doses of 5 or 100 mg DEHA, as a component of a roll-on deodorant, were applied to

samples of human breast tissue. After 24 hours of continuous application, the total amount of

DEHA residing in the skin depot, as well as the amount found in skin washes and the upper and

lower diffusion chambers, was measured (Zhou et al., 2013). Only a small fraction (< 0.05%) of

applied DHEA was found to have passed through the skin samples, with an additional 28% (low

dose) to 34% (high dose) remaining within the skin samples. This finding is consistent with the

prediction of low skin penetration and high retention within the skin. No experimental distinction

was made between stratum corneum and deeper skin layers. It is noteworthy that mass balance

analysis showed only 56 to 81% of the initial amount of DEHA applied was accounted for at the

experimental conclusion (Zhou et al., 2013; ECHA, 2018).

Based on absorption rates from animal studies, CPSC estimated that transdermal absorption rates

for DEHA in animals may be 5- to 10-fold greater than in adult human skin (Wester and

Maibach, 1983 as cited by CPSC, 2014). Hence, it is assumed that adult human skin is 7-fold

less permeable and infant skin 2-fold less permeable than rodent skin (Wormuth et al., 2006 as

cited by CPSC, 2014). It is noteworthy, however, that this estimation is only valid if the

absorption kinetics (the rate at which substances diffuses across the skin to reach the blood

stream) exceed the dose rate (the mass load applied to an area of skin per time (mass/area*time)

(Kissel, 2011). If the dose rate in the animal studies exceed the sorptive capacity of the skin, then

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absorption will be saturated, in which case percutaneous absorption in humans could be greatly

underestimated (CPSC, 2014).

Based on the physical and chemical properties of DEHA (low vapor pressure, high molecular

weight, high Kow, and low water solubility), inhalation of DEHA is not likely unless liquid

containing DEHA is aerosolized. No inhalation studies of DEHA toxicokinetics were identified.

Distribution

Following oral administration, there was no accumulation of DEHA or MEHA in blood, urine or

any other tissue except the stomach.

Metabolism

In humans and in rats, orally administered DEHA is rapidly hydrolyzed to the monoester, mono-

2-ethylhexyl adipate (MEHA) and adipic acid (AA). In homogenates prepared from tissues of

male Wistar rats, the rate of formation of AA from DEHA was approximately the same for all

tissues, whereas the appearance of MEHA was rapid only with pancreatic tissue, and was

negligible in the intestine. These in vitro results are consistent with in vivo Wistar rat studies

where animals were administered a single gavage dose (in corn oil) of 500 mg/kg. Following oral

administration, there was no accumulation of DEHA or MEHA in blood, urine or any other

tissue (except the stomach). The absence of MEHA, the authors concluded, suggests that MEHA

is hydrolyzed more quickly than DEHA. Subsequent in vitro studies using homogenates of rat

liver, pancreas and small intestinal tissue, confirmed that hydrolysis of the monoester (MEHA)

to AA is indeed more rapid than hydrolysis of DEHA to AA. The study authors concluded that a

significant pre-systemic hydrolysis of DEHA occurs in gastrointestinal tissue (Takahashi et al.,

1981).

The metabolism of DEHA was investigated in six male volunteers, who each received a gelatin

capsule of 46 mg deuterium-labeled DEHA formulated in corn oil. No volunteer showed any

adverse effect and no significant changes in biochemical or hematological parameters were

detected. Oxidative metabolites, not the parent DEHA, were identified in the plasma of the

subjects. In the plasma, these metabolites consisted of 2-ethylhexanoic acid (2EHA). No effort

was made to detect AA in the plasma or urine due to loss of the radiolabel. In the urine, the

dominant metabolite was also 2EHA. It was present primarily in a conjugated form, and

accounted for 8.5% of the dose. Oxidation products accounted for 3.5% of the administered dose

(Loftus et al., 1993).

In a more recent study, Silva et al. (2013) evaluated the in vitro metabolism of DEHA using

human liver microsomes. This study identified AA as the major metabolite, along with MEHA

mono-2-ethylhydroxyhexyl adipate (MEHHA) and mono-2-ethyloxohexyl adipate (MEOHA),

which were formed at concentrations of 1/10 to 1/1000 of adipic acid. The authors concluded

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that first DEHA metabolite formed is the hydrolytic monoester MEHA, which is rapidly

hydrolyzed. AA was the major metabolite of DEHA/MEHA (Silva et al., 2013)).

Elimination

The data indicate that there is little, if any, prolonged retention of DEHA or its metabolites in

blood and tissue after oral administration in rodents or humans. In male Wistar rats, urinary

excretion is the dominant route of elimination, followed by breath. The amount excreted in the

feces was characterized as small. By 6 hours following oral administration of a single 500 mg/kg

dose of 14C-labeled DEHA, approximately 19% of the administered dose appeared in urine, with

the largest fraction eliminated between 12 and 24 hours after dosing. Quantified DEHA

metabolites together accounted for approximately 74% of the administered dose excreted in the

urine within 24 hours of administration (Takahashi et al., 1981).

5 Hazard Information2

5.1 Acute Single Dose Toxicity

5.1.1 Acute Oral Toxicity

Lethality of DEHA by acute exposure is low by all routes. Smyth et al. (1951) determined a

median LD50 value of 9110 mg/kg for DEHA from a single-dose oral range-finding study in rats,

with a 14 day post-dose observation period. This report is limited by the absence of information

on the rat strain or proportion of animals of each sex in the treatment groups. ECHA (2018)

reported an unidentified non-GLP OECD Guideline-equivalent study (1955) in which variable

numbers of male and female rats (strain unspecified) were orally administered DEHA without

vehicle (method not described). This study identified a LD50 of about 19,100 mg/kg. It should be

noted that nearly all animals in the LD50 (20.7 mL/kg) treatment group, as well as dose levels

bounding this dose (16 mL/kg and 25 mL/kg) were female (14:1 female), and all of the 5 rats

tested at the LD50 were female. This means that it is not clear whether the calculated LD50 also

applies to males. NTP (1982) estimated LD50 values of 45,000 and 24,600 mg/kg in male and

female F344 rats, respectively, that were given a single bolus gavage dose of DEHA in corn oil

at levels ranging from 80 to 20,000 mg/kg (5/dose/sex) and observed for 14 days. Similar

experiments in B6C3F1 mice yielded LD50 estimates of 15,000 mg/kg in males and 24,600

mg/kg in females (NTP, 1982). Effects on endpoints other than mortality were not reported in

any of these studies.

2 Where available, this report provides significance level p values in all sections. However, source secondary

references often report only that a change was significant without reporting the p level. If no p level is reported in

this text, the p level was not available in the cited secondary reference, but the significance is presumed to be

statistical.

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5.1.2 Acute Dermal Toxicity

A single-dose dermal range-finding LD50 value of 16,300 mg/kg was determined for DEHA in

rabbits observed for 14 days (Smyth et al., 1951). Information on the dermal exposure conditions

in this study was not available. A similar LD50 of > 8670 mg/kg bw was reported for rabbits by

NICNAS without details (OECD, 2005 as cited by NICNAS, 20113).

5.1.3 Acute Inhalation Toxicity

Acute inhalation data for DEHA were limited to one study that found no mortality among rats

exposed for 8 hours to air saturated with DEHA vapor (Smyth et al., 1951). In an GLP-compliant

1998 study cited by ECHA (2018), no mortality was observed in male and female Wistar rats

exposed to 5.7 mg/L DEHA aerosol (mass median aerodynamic diameter [MMAD] = 1.4 µm for

4 hours; observation continued for 14 days after the exposure. During the administration period

and for 5 days post exposure, irregular and accelerated respiration was observed, as well as

attempts to escape and piloerection. No changes in body weight or macroscopic pathological

findings were observed at the end of the study (ECHA, 2018).

5.1.4 Irritation/Sensitization

Existing evidence from rabbit studies supports the conclusion that DEHA is minimally irritating

to skin and eyes. In an unpublished study, rabbits receiving a single application of DEHA to

intact or abraded skin in doses of 3600 - 8700 mg/kg under occlusive conditions for 24 hours

showed dose-related transient mild skin irritation (slight erythema), but no systemic effects, as

evaluated by clinical signs, body weight, food consumption, hematology and urinalysis during

the following 14 days (CTFA, 1967).

A number of unpublished studies tested the dermal irritation and sensitization potential of DEHA

in animals and humans; these have been evaluated in an authoritative assessment of the safety of

DEHA as a cosmetic ingredient (Anonymous, 1984 as cited in Versar, 2010). In rabbits, primary

dermal irritation studies of DEHA alone or in cosmetic formulations, as well as clinical patch

tests of cosmetic formulations containing up to 9.0% DEHA in humans (including a 21-day

cumulative irritancy test), indicated that DEHA is, at most, a weak skin irritant. The human patch

tests of cosmetic products containing DEHA, as well as a study of unformulated DEHA in

guinea pigs, also showed no induction of skin sensitization. Additionally, dermal phototoxicity

tests of DEHA in humans and rabbits showed no phototoxic (primary irritant) or photoallergic

reactions.

3Note: The NICNAS citation does not link to the correct chemical, and it is not clear whether this is really a second

study or an alternative reporting of the Smyth et al. (1951) study.

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Limited eye irritation data are available. In one study that is minimally described, a 0.1 mL of

DEHA (concentration and vehicle not specified) was instilled into one eye of six albino rabbits,

followed by a 72 hour observation period. No irritation was observed at any timepoint (ECHA,

2018).

Dermal sensitization potential of DEHA was evaluated in 10 male guinea pigs using the Draize

test (GLP compliance unknown). On study Day 1, 0.05 mL of a 0.1% solution of DEHA in olive

oil was administered by intracutaneous injection to the shaved back or side skin. Subsequently,

0.1 mL of a 0.1% DEHA solution was injected every other day for a total of 10 injections.

Twenty-four hours after each injection, injection sites were examined for changes including

height and color. Two weeks after the last injection, animals were challenged intradermally with

0.05 mL of the 0.1% DEHA solution. The dermal reaction 24 hours following the challenge

injection was compared with an average of the original 10 induction scores. The area and height

of the retest area was smaller and lower than the average induction reactions. It is concluded that

DEHA is not sensitizing (ECHA, 2018, citing BUA, 1996). A separate study also concluded that

DEHA is not sensitizing in rabbits. In this minimally described study, rabbits were induced with

a single dermal injection of 100% DEHA (in mineral oil) and challenged two weeks later

(Mallette and von Haam, 1952 as cited by ECHA, 2018).

5.2 Repeated Dose Toxicity

A number of repeated-dose oral studies of DEHA have been conducted in rats and mice with a

primary purpose of investigating peroxisome proliferation in the liver, particularly mechanisms

by which it can lead to the formation of hepatocellular tumors. Most of these studies were

conducted in rats exposed to DEHA in the diet for 1-4 weeks at one exposure level in the range

of 1 - 2.5% (10,000 - 25,000 ppm), i.e., at dietary concentrations comparable to those tested in

the NTP (1982) chronic bioassay of DEHA in rats and mice and found to be hepatocarcinogenic

in mice (see Section 5.4). A few of the studies tested mice at longer exposure durations (up to 13

weeks), multiple dietary exposure levels (ranging as low as 1500 ppm) and/or gavage exposure.

As discussed by Versar (2010), effects induced by DEHA in these studies are consistent with

those of di(2-ethylhexyl)phthalate (DEHP) and other hepatic peroxisome proliferators in rats and

mice (Cattley et al., 1998; Chevalier and Roberts, 1998; Doull et al., 1999; IARC 2000a, 2000b;

Lake, 1995). These effects include liver enlargement due to hepatocellular hypertrophy and

proliferation, increased replicative DNA synthesis, increased number and size of peroxisomes

(ultrastructural effects), induction of peroxisomal and microsomal fatty acid-oxidizing enzymes,

alterations in hepatic lipid metabolism including inhibition of cholesterolgenesis, and reduced

serum/plasma cholesterol and triglyceride levels (Barber et al., 1987; Bell, 1983, 1984; Katoh et

al., 1984; Kawashima et al., 1983a, 1983b; Keith et al., 1992; Lake et al., 1997; Moody and

Reddy, 1978, 1982; Reddy et al., 1986; Takagi et al., 1990, 1992; Tomaszewski et al., 1986;

Yanagita et al., 1987). Peroxisome proliferation is a rodent-specific effect that is of questionable

relevance to hazard characterization for humans (Cattley et al., 1998; Chevalier and Roberts,

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1998; Doull et al., 1999; IARC, 2000a; Klaunig et al. 2003; Lake, 1995; Melnick 2001), as

discussed further in Section 5.9.

The National Toxicology Program (NTP) evaluated DEHA for systemic toxicity in 14- and 91-

day oral feeding studies of Fischer 344 rats and B6C3F1 mice (NTP, 1982). In the 14-day study,

male and female Fischer 344 rats and male and female B6C3F1 mice (5/sex/dose) were fed diets

containing 3100 – 50,000 ppm DEHA (males) or 6300 – 100,000 ppm for 14 days. All of the rats

survived, aside from one high-dose female. Reduced food consumption (by an unspecified

amount) and decreased weight gain relative to controls was observed at 50,000 ppm in males and

50,000 ppm and above in females. Females treated with 6300 or 25,000 ppm also had weight

gain decreased by more than 10% relative to controls. Among the mice, the only deaths were at

100,000 ppm; none of the mice survived at this dose. Weight gains decreased by more than 10%

relative to controls were observed in male and female mice fed diets containing 12,500 ppm and

above. The only endpoints evaluated were survival, body weight and food consumption, and the

changes in body weight were often not clearly dose-related, and so no effect levels are identified

from this study.

In a 91-day mouse study (NTP, 1982), mice were administered DEHA in diet at doses of 0,

1600, 3100, 6300, 12,500 or 25,000 ppm (calculated by U.S. EPA, 1992 to correspond to

approximately 0, 400, 700, 1300, 2800 and 7000 mg/kg-day). Decreased weight gain was

observed at several doses, but there was large variability among doses and no dose-response, and

so an effect level for decreased body weight cannot be clearly established from this study.

In the NTP (1982) study, DEHA was also administered to Fisher 344 rats in the diet for 91 days

at concentrations of 0, 1600, 3100, 6300, 12,500, or 25,000 ppm (calculated by U.S. EPA, 1992

to correspond to approximately 0, 100, 200, 400, 700, or 1500 mg/kg-day). Identification of an

effect level is difficult, in the absence of a clear dose-response, but decreased body weight gain

of 10% or more was reported for male rats at 12,500 ppm in feed and higher (~ 700 mg/kg). In

females, decreased weight gain was reported at the two top doses, but were 5.7% and 8.2%,

respectively. Food consumption was not decreased. Because body weight and survival were the

only endpoints evaluated, these 91-day studies are not adequate to identify a clear NOAEL.

Several studies evaluated systemic endpoints as part of an evaluation of reproductive toxicity. In

Fischer 344 rats exposed to 1570 mg/kg-day of DEHA (25,000 ppm) in the diet for 4 weeks,

Kang et al. reported a 50% increase in relative liver weight and a 10% decrease in body weight

in males. However, no effects on serum markers of hepatotoxicity (e.g., Alanine transaminase

[ALT], Aspartate transaminase [AST], Gamma-glutamyl transpeptidase [GGT]), or histological

effects were observed. No hepatic effects were observed at a calculated dose of 318 mg/kg-day

(Kang et al., 2006, as cited by CPSC, 2014). The high dose of 1570 mg/kg-day was considered

adverse, in light of the magnitude of the liver weight change and decreased body weight.

In a 28 day study, male and female Crj:CD (SD) rats (10/sex/dose) were given DEHA in corn oil

by gavage at dose levels of 0, 40, 200 or 1000 mg/kg-day for at least 28 days (Miyata et al.,

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2006). In addition to the reproductive endpoints described in Section 5.5, evaluations included

hematology, serum biochemistry, serum hormones (thyroid stimulating hormone [TSH], T3, T4,

and reproductive hormones), and weight and histopathology of reproductive organs, endocrine-

related organs, and several other major organs in both sexes.

There were no treatment-related effects on body weight. Relative liver weights were

significantly increased (~20%; p<0.01) at 1000 mg/kg-day in both sexes, but without

accompanying serum chemistry or histopathology changes. In light of the peroxisome

proliferative activity of DEHA, the increased liver weight at 1000 mg/kg-day is considered

potentially adverse even though no histopathological findings were reported. The authors

identified a NOAEL for liver effects of 200 mg/kg-day (Miyata et al., 2006). Relative kidney

weights were significantly (p<0.01 in males and p<0.05 in females) increased in both sexes at the

high dose, and in males at the mid dose (p<0.01). Increased eosinophilic bodies and hyaline

droplets were seen at the high dose in males, but not in any mid-dose males. More importantly,

increased kidney weights were observed in mid-dose males in the absence of increased hyaline

droplets, and in female rats at the high dose. Hyaline droplets in male rats are suggestive of male

rat-related alpha-2u-globulin nephropathy, but there was no specific staining for this protein, so

an association with this protein could not be verified. The observation of increased kidney

weight in the absence of hyaline droplets and the increased female kidney weight together

suggest that at least some of the increased kidney weight is due to some cause other than alpha-

2u-globulin nephropathy. Increased kidney weights are considered adverse and relevant to

humans. The NOAEL for increased kidney weight was therefore 40 mg/kg-day in males and 200

mg/kg-day in females. Significantly increased relative adrenal weight was also seen in females

(p<0.05) at the high dose, resulting in a NOAEL for endocrine effects at 200 mg/kg-day in

females; the NOAEL for endocrine effects in males was 1000 mg/kg-day.

5.3 Chronic Toxicity/Carcinogenicity

F344 rats (50/sex/dose) and B6C3F1 mice (50/sex/dose) were fed a diet containing 0, 12,000 or

25,000 ppm DEHA for 103 weeks and observed for an additional 1-3 weeks following the end of

exposure (NTP, 1982). Clinical signs, survival, body weight, gross pathology, and

histopathology of major tissues and organs and all gross lesions were evaluated.

Based on U.S. EPA (1988) reference values for food consumption and body weight for chronic

exposure in F344 rats, estimated doses of DEHA in rats were 0, 948 and 1975 mg/kg-day for the

males and 1104 and 2300 mg/kg-day for the females4 (NTP, 1982 did not report food

consumption). Mean body weights of the high-dose male and female rats were reduced

4 Based on a food factor of 0.079 for male F344 rats in a chronic study and 0.092 for female F344 rats in a chronic

study. This conversion factor was used in previous CPSC reports (Versar, 2010; CPSC, 2014). Other authoritative

reviews and secondary sources used other conversions. For example, the doses were reported as 600 and 1250

mg/kg-day by ECHA (2011), as 700 and 1500 mg/kg-day by U.S. EPA (1992) for its RfD, and as 697 and 1509

mg/kg-day for males and 860 and 1674 mg/kg-day for female rats by U.S. EPA (1991) for its cancer assessment.

15

throughout the study by more than 10% (as estimated from graphical data). At the end of the

exposure period, the mean body weights of the high-dose males and females were approximately

12 and 22% lower than controls, respectively (as estimated from growth curves). No neoplastic

or non-neoplastic lesions or other compound-related adverse effects were observed in dosed rats.

The high dose (1975 mg/kg-day for males and 2300 mg/kg-day for females) can be considered a

LOAEL based on decreased body weight. The low dose of 948 mg/kg-day for males and 1104

mg/kg-day for females can be considered a NOAEL.

The mouse study (NTP, 1982) tested dietary DEHA concentrations of 0; 12,000; and 25,000

ppm. These correspond to estimated doses of 0, 2040 and 4250 mg/kg-day for both sexes, based

on U.S. EPA (1988) reference values for food consumption and body weight5. Mean body

weights of low- and high-dose male and female mice were lower than controls throughout the

study and the decreases were dose-related. In males, there was substantial variability in the

control body weight throughout the study, and so it is not clear whether the trend in the low-dose

group was biologically significant; the decrease at the high dose was >10%. In female mice, the

decrease was >20% compared to controls at both doses. Survival at the end of study in the

control, low-dose and high-dose groups was 72, 64 and 82% in males and 84, 78 and 73% in

females. There were no treatment-related non-neoplastic lesions or clinical signs of toxicity at

either dose. Thus, the low dose of 2040 mg/kg-day was a NOAEL in males and a LOAEL in

females, based on decreased terminal body weight compared to controls.

Liver tumors (hepatocellular carcinomas and adenomas combined) were induced in both sexes.

As shown in Table 2, incidences of combined hepatocellular tumors were significantly increased

in high-dose male mice and low- and high-dose female mice. The increase was dose-related in

males, and statistically significant in pairwise comparisons (see Table 2 for significance levels).

In comparison, the historical control incidence in male mice was 22% (range 14-30%), and in

female mice was 8% (range 2-20%). Time-to-tumor analysis of the data for the female mice

showed that tumor development in the dosed groups was significantly shorter (p=0.002) relative

to the control group, whereas time-to-tumor analysis in high-dose males was not significantly

different. No compound-related non-neoplastic lesions were observed in the liver or other

tissues.

As discussed further in the context of mode of action (MOA), the mouse liver tumors were

considered related to PPAR alpha (Lake et al., 1997), and thus not relevant to humans (Felter et

al., 2018).

5 Based on a food factor of 0.17 for both sexes. This conversion factor was used in previous CPSC reports (Versar,

2010; CPSC, 2014). Doses reported by other authoritative reviews included 1715 and 3570 mg/kg-day (ECHA,

2018), 2800 and 7000 mg/kg-day (U.S. EPA 1992, in its RfD), and 2659 and 6447 mg/kg-day for male mice and

3222 and 8623 mg/kg-day for female mice (U.S. EPA, 1991, in the cancer assessment).

16

Table 2. Liver Tumor Incidence in DEHA Treated Micea

Dose

mg/kg-day

(ppm in feed)

Hepatocellular Adenoma or Carcinoma

Males Females

0 13/50

(26%)

3/50

(6%)

2040

(12,000)

20/49

(41%)

19/50c

(38%)

4250

(25,000)

27/49b

(56%)

18/49c

(39%) aNTP (1982) bSignificantly different from control at p=0.003 cSignificantly different from control at p<0.001

Carcinogenicity results of chronic feeding studies of DEHA in rats and dogs were briefly

reported by Hodge et al. (1966), but without sufficient documentation. No compound-related

tumors were induced in rats exposed to 0, 0.1, 0.5 or 2.5% (1000, 5000 or 25,000 ppm) DEHA in

the diet for 2 years. These negative results are consistent with those of the NTP (1982) rat study

summarized above, which also tested DEHA in dietary concentrations up to 25,000 ppm. In the

same study, no tumors were found in dogs exposed to 0, 0.07, 0.15 or 0.2% (700, 1500 or 2000

ppm) DEHA in the diet for 1 year.

In other carcinogenesis studies conducted by Hodge et al. (1966), C3H/AnF mice (50/sex/dose)

were exposed to DEHA by dermal application or subcutaneous injection. In the dermal portion

of this study, weekly application of 0.1 or 10 mg of DEHA in acetone to a clipped area of back

skin under non-occlusive conditions for life caused no gross or histological evidence of tumor

formation at the application site. In the subcutaneous portion of the study, a single 10 mg dose of

DEHA caused no injection site tumors following lifetime observation. The author of this report

considers these studies to be minimally informative with regard to carcinogenicity in mice,

because tumors were evaluated only at the application site (dermal) or injection site

(subcutaneous exposure).

17

5.4 Reproductive Toxicity

DEHA has been suspected of having effects on the male reproductive system because it shares

similarities in chemical structure and metabolism with DEHP, a well-documented inducer of

testicular toxicity and antiandrogenic effects in rats and other laboratory animals (SCENIHR,

2007; IARC, 2000b). Young animals are much more sensitive to DEHP testicular toxicity than

adults, and male rats have been shown to be particularly susceptible to antiandrogenic effects of

DEHP when exposed during the perinatal period (NTP-CERHR, 2005). In contrast to DEHP,

however, DEHA does not induce any adverse reproductive effects in male rats exposed

perinatally, or exposed beginning as young adults (5-11 weeks) for 4, 13 or 103 weeks (Dalgaard

et al., 2002, 2003; Kang et al., 2006; Miyata et al., 2006; Nabae et al., 2006, NTP, 1982).

A GLP-compliant (OECD Guideline 415) 1-generation reproductive toxicity study is available

(CEFIC, 1988; ICI, 1988b, as described by ECHA, 2018; U.S. EPA, 1992; OECD, 2000). In this

study, male and female Wistar rats (30 females and 15 males/dose) were administered DEHA at

0, 300, 1800, or 12,000 ppm in the diet. The males were exposed for 10 weeks premating and

during mating, and the females were exposed for 10 weeks prior to mating, through mating and

gestation, until the end of lactation (postnatal day; PND22). The offspring were reared to PND

36. Based on the companion developmental toxicity study, doses were 0, 28, 170 or 1080 mg/kg-

day (U.S. EPA, 1992; ECHA, 2018). Histopathology evaluation for rats in the study was limited

to the reproductive tissues and abnormal tissues.

In the study, there were no clinical signs of toxicity or changes in body weight or feed

consumption during the premating period, and no effects on male or female fertility were

observed. Adverse effects were limited to changes in body weight and liver weight at the high

dose. Maternal body weight gain during gestation was described as being “marginally” reduced

at the high dose, with the changes being statistically significant for a few treatment intervals.

Litter size was slightly, but not significantly, reduced at the high dose, but the number of live

born pups was not affected. This small change in litter size was considered incidental by the

author. Mean pup weight was unaffected on PND1, but pup weight gain and total litter weight

were reduced throughout the whole of the post-partum phase at the high dose. ECHA (2018)

considered this decrease to be secondary to the decreased maternal weight gain. Although it is

possible that there were effects on lactation (maternal weight was not recorded during lactation),

the pup weight changes could be due to a direct effect, given that there was no effect on pup

weight on PND1. Sporadic whole litter losses (total of 4) were noted in all exposed groups and

not the controls, but were not considered treatment-related, because the incidence was low and

not dose-related.

Postmortem examinations of the parental animals, conducted in males at the end of the mating

period and females after weaning of the offspring, showed increased absolute and relative liver

weights in both sexes at 1080 mg/kg-day. No exposure-related histopathological changes

occurred in the reproductive tissues of the parental males and females (including those that failed

to breed successfully), and no exposure-related gross pathologic changes occurred in the

offspring. The high-dose of 1080 mg/kg-day was a systemic LOAEL, based on reduced maternal

body weight gain during gestation, increased liver weight (considered to be associated with

18

peroxisome proliferation), and reduced pup weight gain that may have been secondary to the

maternal effect. The maternal and developmental NOAEL was 170 mg/kg-day and is the basis of

the U.S. EPA (1992) oral RfD together with the ICI (1988a) developmental toxicity study.

ECHA (2018) also derived its DNEL of 170 mg/kg-day for the general population based on this

study. The reproductive NOAEL was 1080 mg/kg-day.

In an enhanced screening assay (OECD Guideline 407), both male and female reproductive

endpoints were assessed in 8-week-old Crj:CD (SD) rats (10/sex/dose) given DEHA in corn oil

by gavage at dose levels of 0, 40, 200 or 1000 mg/kg-day for at least 28 (Miyata et al., 2006).

Systemic effects in this study were described in Section 5.3. Males were sacrificed on day 29 and

females were sacrificed in the diestrus stage on days 30-34. Evaluations included estrus cycling

in females (assessed daily from day 22 until the day of sacrifice), sperm morphology and number

in males, serum hormones (thyroid stimulating hormone [TSH], T3, T4, testosterone, follicle-

stimulating hormone [FSH], luteinizing hormone [LH] and estradiol) and weight and

histopathology of reproductive organs and endocrine-related organs, and several other major

organs in both sexes. There was no effect on body weight. Reproductive effects were not

observed in male rats. Ovarian follicle atresia (absence or disappearance by degeneration) was

observed in 4/10 females at 1000 mg/kg-day (compared to 0/10, 0/10 and 0/9 female rats at 0, 40

and 200 mg/kg-day). Two of the four rats with ovarian follicular atresia had a prolonged estrus

cycle (estrous stage durations of 4 and 10 days). Although the sample size was relatively small in

this study, and there were no effects on hormone levels, these effects are treatment-related, in

light of the clean background and clear difference from the background data. In addition, the

prolonged estrous stage was associated with histopathological changes in the ovary. Thus, results

suggest a NOAEL of 200 mg/kg-day and LOAEL of 1000 mg/kg-day for reproductive toxicity in

female rats. A NOAEL of 1000 mg/kg-day and no LOAEL was identified for male reproductive

toxicity in rats.

Wato et al. (2009) evaluated potential ovarian toxicity of DEHA. In a set of repeat-dose toxicity

studies, DEHA was administered by gavage for 2 or 4 weeks to 6 week old Crl:CD(SD) female

rats (10/dose) at doses of 0, 200, 1000 or 2000 mg/kg-day. A significant (p<0.01 to 0.05)

decrease in relative ovary weight was observed at 2000 mg/kg-day; this effect was considered

attributable to decreased corpus luteum formation. Increased large follicle atresia was observed

at 1000 mg/kg-day and above following 2 and 4 weeks of dosing. General toxicological effects

in treated dams included significant (p<0.01 to <0.001) increases in relative liver and kidney

weights following 2- and 4-week dosings at doses of ≥1000 mg/kg-day. Red staining around the

perineum was reported at 2000 mg/kg-day in the 2-week study and at 1000 mg/kg-day and above

in the 4-week study, but was not additionally discussed by the study authors. A significant (p <

0.05) decrease was observed in the mean length of the estrous cycle at the 200 mg/kg-day dose

group in the 4-week study. This effect was not dose related, however, as no decrease was

observed in the 1000 and 2000 mg/kg-day dose groups following either 2 or 4 weeks of dosing.

The study authors identified a NOAEL 200 mg/kg based on ovarian effects following both 2 and

4 weeks of dosing.

19

Wato et al. (2009) also conducted a separate female fertility and developmental toxicity study. In

this study, Crl:CD(SD) female rats were gavage dosed with 0, 200, 1000 or 2000 mg/kg-day for

2 weeks before mating, throughout mating and until gestation day (GD) 7. Reproductive effects

identified by Wato et al. included a significant increase in mean estrus length (≥1000 mg/kg-day,

p<0.05), an increase in the post-implantation loss rate (1000 mg/kg-day, p<0.05), a decrease in

the number of live embryos (p<0.05), and an increase in the pre-implantation loss (p<0.01) at

2000 mg/kg-day. Large follicle atresia, decreased corpus luteum formation and increased

follicular cysts were also observed at doses of 1000 mg/kg-day and above. General maternal

dose-related effects included staining around perineum (≥ 1000 mg/kg-day) and a significant

decrease in body weight and body weight gain prior to the mating period (2000 mg/kg-day, p<

0.05), but not during gestation. Based on these data, the study authors identified a NOAEL of

1000 mg/kg for general toxicity in dams, and a NOAEL of 200 mg/kg-day for reproductive

functions of dams and early embryonic development.

In other subchronic and chronic studies, no histopathological effects were observed in the

reproductive organs (testes, seminal vesicles, prostate, ovary or uterus) of male or female F344

rats or B6C3F1 mice exposed to DEHA in the diet as part of general systemic toxicity studies at

concentrations as high as 25,000 ppm for 13 or 103 weeks (NTP, 1982). The corresponding

doses were ~1500 mg/kg-day in rats and ~7000 mg/kg-day in mice for the subchronic study, and

~2000 mg/kg-day in rats and ~4250 mg/kg-day in mice for the chronic study.

Nabae et al. (2006) and Kang et al. (2006) both investigated the testicular toxicity of DEHA in

greater detail. In each study, 11-week-old male F344 rats (6/dose) were exposed to DEHA in the

diet at concentrations of 0, 6000 or 25,000 ppm for 4 weeks. Nabae et al. (2006) reported

average intakes of 0, 318 and 1570 mg/kg-day. Evaluations included body weight,

spermatogenesis (sperm number, motility and morphology abnormalities), and relative weight

and histopathology of the testes, epididymes, prostate and seminal vesicles. Both studies reported

significantly reduced terminal body weight (>10%, p<0.01) at 1570 mg/kg-day. Significantly

increased relative testes weight was also reported by Nabae et al. (2006) at 1570 mg/kg-day

(p<0.05; 9.3% higher than controls).The author did not considered this change adverse because

relative testes weight was increased rather than decreased (possibly secondary to reduced body

weight) and not accompanied by abnormal spermatogenesis or testicular histopathology findings.

Additionally, this effect was not induced by the same DEHA exposure in the Kang et al. (2006)

study. Additional experiments by Kang et al. (2006) to evaluate the interaction of testicular and

liver toxicity showed that similar DEHA exposures did not cause testicular toxicity in rats that

were pretreated with thioacetamide to induce liver damage. In contrast, DEHP (25,000 ppm for

4 weeks) caused testicular toxicity (e.g., seminiferous tubule atrophy and degeneration) that was

enhanced by liver damage induced by thioacetamide. Additional experiments by Nabae et al.

(2006) demonstrated that DEHA exposures did not cause testicular toxicity in rats that were

pretreated with five consecutive weekly subcutaneous injections of folic acid to induce chronic

20

renal dysfunction6. This was in contrast to rats treated with DEHP (25,000 ppm for 4 weeks),

which caused testicular toxicity (e.g., decreased testicular weights, seminiferous tubule atrophy

and diminished sperm counts) that was enhanced under conditions of renal dysfunction induced

by folic acid. The high dose of 1570 mg/kg-day was a NOAEL for male reproductive toxicity of

DEHA in these studies.

No multi-generation reproductive toxicity study of DEHA was located.

5.5 Prenatal, Perinatal, and Postnatal Toxicity

Dalgaard et al. (2002, 2003) conducted two studies in Wistar rats to investigate the

developmental effects of prenatal and postnatal DEHA exposures; a smaller dose range-finding

study (8 dams/dose) and a main study (20 dams/dose). In the range-finding study, dams were

administered DEHA by gavage at dose levels of 0, 800 or 1200 mg/kg-day from GD 7 to PND

17. Evaluations included maternal clinical signs and body weight during the dosing period,

pregnancy length, number and size of litters, sex distribution, body weight of pups at birth and

on PND 3, postnatal survival through PND 21, anogenital distance on PND 3 and areola/nipple

retention on PND 13 in male pups, and weights of testes, epididymides, ventral prostate and

seminal vesicles in male pups on PND 21. Statistically significant effects included decreased

maternal body weight gain during GD 7-21 (p<0.05), increased pregnancy length (p<0.01), and

increased percentage of perinatal loss (defined as (number of implantations – live pups at

weaning)/number of implantations) at 1200 mg/kg-day (p<0.05). Body weights of male and

female pups were significantly decreased at birth at 1200 mg/kg-day (p<0.05) and on PND 3

(only PND evaluated) at ≥800 mg/kg-day (p< 0.01). The study found no antiandrogenic effects,

but identified a LOAEL of 800 mg/kg-day and no NOAEL for developmental toxicity in rats

based on decreased pup body weight. Maternal effects were reported only at 1200 mg/kg-day.

In the main study of perinatally Wistar exposed rats, dams (20/dose) were administered DEHA

by gavage in peanut oil at dose levels of 0, 200, 400 or 800 mg/kg-day from GD 7 to PND 17

(Dalgaard et al., 2002, 2003). Evaluations included the endpoints assessed in the range finding

study, as well as additional endpoints for onset of sexual maturation in both sexes, levels of

reproductive hormones in males, sperm quality, weight and histopathology of male reproductive

organs, and other organ weights. For analyses of sexual maturation, hormones and sperm quality,

one male and one female from each litter were retained until adulthood. Statistically significant

effects included increased gestation length at 800 mg/kg-day (p<0.01), decreased body weight of

male and female pups at birth (p<0.05) and on postnatal day 3 (p<0.01) at 800 mg/kg-day, and a

dose-related decrease in pup survival at ≥400 mg/kg-day (p<0.01). No androgenic endpoints

were affected. Relative liver weight was significantly increased in male offspring on PND 21 at

800 mg/kg-day (p<0.05) but not as adults. The only statistically significant (p<0.05) changes in

adult male offspring were decreased body and adrenal weights at 800 mg/kg-day. The study

6 These experiments were conducted to investigate the potential for an interaction with folate, due either to impaired

clearance due to an effect of folic acid on renal function, or a reproductive effect of folic acid via modification of

zinc absorption.

21

identified a NOAEL of 200 mg/kg-day and LOAEL of 400 mg/kg-day for developmental

toxicity in rats based on the increased postnatal deaths. The maternal NOAEL was 400 mg/kg-

day, based on increased gestation length at a LOAEL of 800 mg/kg-day.

In an unpublished GLP-compliant developmental toxicity study, Wistar-derived female rats

(24/dose) were fed diets7 containing 0, 300, 1800 or 12,000 ppm DEHA on GD 1-22 (ICI, 1988a,

as cited by Versar, 2010; ECHA, 2018). Average intake of DEHA was reported to be 0, 28, 170

or 1080 mg/kg-day. Maternal evaluations included clinical observations, body weight and food

consumption throughout the study, and gross pathology following sacrifice on GD 22.

Developmental endpoints evaluated included gravid uterus, litter and fetal weights, and numbers

of corpora lutea, implantations (early and late intra-uterine deaths) and live fetuses. All fetuses

were examined for gender, cleft palate, and external, visceral, skeletal and macroscopic brain

abnormalities. Maternal effects occurred at 1080 mg/kg-day and consisted of statistically

significant reductions in food consumption and body weight gain (-13%) throughout gestation.

Fetal effects were observed at ≥170 mg/kg-day, and included several minor skeletal defects (e.g.,

partially ossified parietals of the skull) and variations indicative of slightly reduced ossification

(e.g., partially ossified transverse process of the 7th cervical vertebrae) and two visceral

variations involving the ureters (kinked ureter, slightly dilated ureter). The authors considered

the ureter variations, as well as the reduced ossification as indicated by the minor skeletal defects

and variations, to be the result of slight fetotoxicity, but ECHA (2018) considered the changes

non-adverse. Based on the authors’ interpretation of the results, this study identified a NOAEL of

28 mg/kg-day and LOAEL of 170 mg/kg-day for prenatal developmental toxicity in rats. EPA

(1992), however, considered the developmental changes at 170 mg/kg-day to be non-adverse and

classified 170 mg/kg-day as a NOAEL, and 1080 mg/kg-day as the LOAEL.

In another unpublished GLP-compliant (OECD Guideline 414, except that dosing was in diet

instead of by gavage) developmental toxicity study, groups of 21-27 pregnant New Zealand

White rabbits were treated with DEHA in the diet on days 6 to 29 post-coitum, at target doses of

0, 40, 80 or 160 mg/kg-day (Anonymous, 2014, as cited by ECHA, 2018). The mean actual

measured intake was 0, 36, 70, and 145 mg/kg-day, although there was substantial inter-

individual variability. The doses were based on a range-finding assay in which pregnant rabbits

were administered DEHA in the diet at 100-1000 mg/kg-day and 300 on post-coitum days 7-29,

in which severe toxicity was observed at 300 mg/kg-day. There was no maternal toxicity, based

on the absence of an effect on clinical observations, body weight changes, food/water

consumption, mortality and effects on ovaries and uterus. There were no toxicologically relevant

effects on litter size, sex ratio, fetal body weight; or external, visceral, or skeletal malformations

or variations. Thus, the high dose of 145 mg/kg-day was a maternal and developmental NOAEL.

DEHA and DEHP have the metabolite 2-ethylhexanol (2-EH) in common. Several studies used

DEHA to investigate the hypothesis that 2-EH is responsible for some of the male reproductive

effects of DEHP. In particular, if 2-EH causes these effects of DEHP, DEHA could

hypothetically augment DEHP-induced changes in male reproductive endpoints when the two

7 Administration in diet was a deviation for test guidelines, which recommend gavage dosing unless otherwise

justified.

22

compounds are administered in combination, even though DEHA does not produce these effects

on its own. In these studies, rats were administered either DEHP (300 or 700 mg/kg-day) or

DEHP (750 mg/kg-day) in combination with DEHA (400 mg/kg-day) by gavage from GD 7 to

PND 17 (Borch et al., 2004, 2005; Jarfelt et al., 2005, as cited by Versar, 2010). Exposure to

DEHA alone was not tested. Examination of fetal, prepubertal and adult male offspring found

that anti-androgenic and testicular effects of DEHP were not modulated by coadministering

DEHA with DEHP. Endpoints evaluated in these studies included weight and histopathology of

reproductive organs, testicular apoptosis, anogenital distance and nipple retention, sperm number

and motility, and reproductive hormones

In a dominant lethal study, Singh et al. (1975) administered a single dose of DEHA by

intraperitoneal (ip.) injection to male Harlan/ICR albino Swiss mice (n=10) at dose levels of 0.5,

1.0, 5.0 or 10.0 mL/kg immediately prior to an 8 week mating period. (The study did not provide

the DEHA concentration; it is presumed to be neat.) The study reported that a single ip. injection

at the highest dose tested significantly (p<0.05) reduced the percentage of pregnancies

throughout the 8-week mating period; no effects were observed at any of the lower doses. An

increased number of early fetal deaths (p<0.1) was observed at the two highest dose levels

throughout the 8 week mating period which showed a significant relationship with dose (p<0.01)

(Singh et al., 1975). A dose level of 922 mg/kg was identified as a NOAEL (OECD, 2000).

5.6 Genotoxicity

DEHA was negative or marginal in a variety of in vitro and in vivo genotoxicity assays.

When tested in vitro, DEHA did not induce gene mutations in Salmonella typhimurium strains

TA98, TA100, TA1535, TA1537 or TA1538 (Seed, 1982; Simmon et al., 1977; Zeiger et al.,

1985), or in mouse lymphoma L5178Y cells in the presence or absence of exogenous metabolic

activation (McGregor et al., 1988). Additionally, urine from rats that were administered daily

gavage doses of 2000 mg/kg-day DEHA for 15 days was not mutagenic to S. typhimurium strains

TA98, TA100, TA1535, TA1537 or TA1538 with or without metabolic activation (DiVincenzo

et al., 1985). DEHA did not induce sister chromatid exchanges, micronuclei or chromosomal

aberrations in cultured rat hepatocytes without exogenous metabolic activation (Galloway et al.,

1987; Reisenbichler and Eckl, 1993). When tested in cultured Chinese hamster ovary (CHO)

cells, DEHA did not induce sister chromatid exchanges with or without metabolic activation,

although chromosomal aberrations were induced in the absence but not presence of metabolic

activation (Galloway et al., 1987). SCENIHR (2016) noted that the CHO chromosome aberration

assay was limited in that it did not report on cytotoxicity. DEHA was inactive in a BALB/c-3T3

cell transformation assay (Matthews et al., 1993). DEHA did not induce unscheduled DNA

synthesis in primary rat hepatocytes incubated with DEHA (unpublished CMA studies, 1982d, as

cited by OECD, 2000).

In in vivo tests, micronuclei were not induced in bone marrow cells from mice that were

administered DEHA doses as high as 2000 mg/kg-day for 3 days by ip. injection (Shelby et al.,

1993). There were also no chromosomal aberrations in bone marrow cells of mice administered

23

a single unspecified ip. dose (Shelby and Witt, 1995). Feeding or injection of DEHA did not

induce sex-linked recessive lethal mutations in Drosophila melanogaster (Woodruff et al., 1985).

Results from a dominant lethal assay in male mice administered a single high dose of DEHA (10

mL/kg) by ip. injection (Singh et al., 1975) have been characterized as positive (Versar, 2010),

“slightly positive” (SCENIHR, 2016), and negative (OECD, 2000). Interpretation of this study is

complicated because the authors did not report standard endpoints of pre- and post-implantation

loss, even though corpora lutea were counted. Unscheduled DNA synthesis was stimulated in

hepatocytes from rats administered a single 3.78 mmol/kg (1401 mg/kg) dose of DEHA by

gavage (Busser and Lutz, 1987) but not from mice gavaged with a single 1000 or 2000 mg/kg

dose of DEHA (Miyagawa et al., 1995).

5.7 Mechanistic Studies

DEHA was evaluated by the U.S. EPA Endocrine Disrupter Screening Program (EDSP) in a

suite of 18 ToxCast estrogen receptor (ER) high‐throughput screening assays. The suite includes

assays that measure ER receptor binding, receptor dimerization, receptor DNA binding, gene

transactivation, transcriptional expression, and cell proliferation. Cumulative suite accuracy was

93% for the 40 in vitro reference chemicals, and 84% to 95% for 43 in vivo reference chemicals

with independently verified results in two or more guideline‐like uterotrophic studies. Based on

the cumulative results of the 18 assay suite, DEHA was identified as inactive for direct estrogen

receptor activity (metabolites and other potential pathways of estrogenic activity were not

evaluated) (Browne et al., 2015).

The “gold standard” for identifying potential estrogen receptor agonists is the OECD-validated

Uterotrophic Bioassay (OECD Test Guideline [TG] 440) (Kleinstreuer et al., 2015). This short-

term in vivo assay is part of the U.S. EPA endocrine disruptor screening program (EDSP) for

evaluating the potential for chemicals to elicit estrogenic activity (OECD, 2007; Kleinstreuer et

al., 2015). The endpoint measured is an increase in uterine weight caused by ER-mediated water

imbibition and cellular proliferation in the uterine tissue. In this assay, 20-day-old immature

female rats were administered DEHA (or a solvent control) subcutaneously on three consecutive

days. The highest dose level was 1000 mg/kg-day, which was the maximum tolerated dose as

determined from preliminary tests. DEHA binding to human ER and ER mediated gene

transactivation were also evaluated. DEHA tested negative for estrogenic activity in all assays.

These data are supported by in vivo receptor-mediated gene activation studies in transgenic mice

where DEHA at doses from 30 to 100 mg/kg-day did not induce ER-mediated gene expression in

any tissue (ter Veld et al., 2008). Likewise, a follow-up study showed that DEHA (100 mg/kg-

day) was unable to elicit ER-mediated gene activation in fetuses of pregnant female mice (ter

Veld et al., 2008). Taken together this battery of short-term in vivo studies, in addition to in vitro

receptor based bioassay results, demonstrate that neither DEHA nor its metabolites, could

mediate estrogenic activity either directly (i.e. by binding to the ER) or indirectly (i.e., by

inhibiting enzymes that metabolize estrogen, or induce estrogen productions). Therefore,

24

together these assays effectively rule out both direct and indirect roles for either DEHA or its

metabolites as estrogenic mediators.

Consistent with these findings, Miyata et al. (2006) showed the DEHA has no binding activity

with the estrogen receptor, as detected in a yeast two-hybrid assay. It is noteworthy, however,

that DEHA is rapidly hydrolyzed to multiple metabolites, especially following oral absorption,

and these metabolites largely remain to be evaluated for reproductive toxicity. Miyata et al.

speculated that ovarian effects observed in reproductive studies may be attributable to effects on

the hypothalamic-pituitary-gonad axis. No mechanistic studies were identified that have

investigated this possibility.

5.8 Mode of Action (MOA)

In rodents, peroxisome proliferation is a well-studied MOA for tumor formation. Peroxisome

proliferators, such as DEHP, cause liver-related changes that include increased liver to body

weight ratios due to hepatocellular hypertrophy and proliferation, increased replicative DNA

synthesis, increased number and size of peroxisomes (ultrastructural effects) and induction of

peroxisomal and microsomal fatty acid-oxidizing enzymes, among other changes. Overall, the

weight of evidence from a large number of studies supports the existence a PPARα-dependent

MOA for liver tumor formation in rodent models (Corton et al., 2014; Felter et al., 2018). As

described by Felter et al. (2018), the key events for this MOA are: 1) activation of PPARα, 2)

alteration of cell growth pathways, 3) alteration in hepatocyte fate including increased cell

proliferation and decreases in apoptosis, and 4) clonal expansion leading to tumors.

Several studies have shown that DEHA induces peroxisome proliferation in rats and mice. The

effects induced by DEHA in rats and mice are consistent with those described for DEHP and

other hepatic peroxisome proliferators, as discussed in Section 5.3 of this assessment. As part of

an investigation of the reasons for the differences between rats and mice in the

hepatocarcinogenic potential of DEHA, Lake et al. (1997) conducted a detailed evaluation of the

hepatic effects of DEHA. They found that DEHA induced a dose-dependent increase in relative

liver weight and hepatic peroxisome proliferation in both species, and the magnitude of induction

peroxisome proliferation was similar in both species. An increased hepatocyte labeling index,

indicative of replicative DNA synthesis, was seen in both species after 1 week of treatment.

However, a marked interspecies difference was observed at longer time periods (4 and 13

weeks). In rats, DEHA did not induce a sustained increase in replicative DNA synthesis at either

of these time points at doses up to 4% in the diet, while increased replicative DNA synthesis was

seen in mice at 1.2% and 2.5% (the highest dose tested) in the diet. These dietary levels of

DEHA are the same as those that caused significant increases in hepatocellular tumors in mice,

but not rats (NTP, 1982).

The data of Lake et al. (1997) are consistent with the hypothesis that the hepatocellular tumors

are related to peroxisome proliferation, and specifically are due to a PPARα (peroxisome

25

proliferation-activated receptor alpha) dependent MOA. Even though PPARα activation in rats

leads to liver tumors for other chemicals, liver tumors do not occur in DEHA-exposed rats,

although they do occur in DEHA-exposed mice (NTP, 1982). In the rats, sustained increased cell

proliferation does not occur, even though the first key event is activated. Furthermore, the doses

at which the sustained cell proliferation was seen in mice are consistent with the dose-response

for the tumors in mice.

The importance of sustained replicative DNA synthesis applies more generally to the relevance

of this MOA to the human liver, which undergoes PPARα activation, but not replicative DNA

synthesis. Furthermore, in humans, activation of PPARα does not lead to increased liver to body

weight ratios, oxidative enzyme induction or other responses typically associated with sustained

PPARα activation observed in wild-type mice (Felter et al., 2018; Ito et al., 2012). For DEHA,

this conclusion is specifically supported by a key study that evaluated transgenic mice expressing

human PPARα (hPPARα). When these mice were compared to wild-type mice expressing

normal mouse PPARα (mPPARα), DEHA was a much weaker activator of hPPARα than mouse

PPARα. Furthermore, DEHA was reported to be much less potent than DEHP at activating

hPPARα (Ito et al., 2012). Although these data support a weak PPARα-mediated MOA for

DEHA-induced hepatotoxicity and liver tumors in rodents, the weight of evidence supports the

conclusion that a PPARα MOA is either “not relevant” or “unlikely to be relevant” in humans

(Felter et al., 2018).

5.9 Lowest Hazard Endpoints by Organ System and Exposure Duration

The primary systemic effects of DEHA are increased liver weight (related to peroxisome

proliferation) and decreased body weight.

Decreased body weight was frequently observed in toxicity studies. Although there was not a

fully consistent pattern of effect levels, effects occurred in the 1500 – 2000 mg/kg-day dose

range, regardless of study duration; some reproductive toxicity studies reported decrements in

maternal body weight at doses as low as 1000 mg/kg-day (Dalgaard et al., 2002, 2003; ICI,

1988b, as cited by ECHA, 2018; U.S. EPA, 1992; OECD, 2000). Biologically significant

decreases in body weight were seen at dietary levels of 25,000 ppm in rats and 12,000 ppm in

female mice (about 2000 mg/kg-day for rats and female mice) exposed for 2 years (NTP, 1982),

but 12,000 ppm (1080 mg/kg-day) in rats in a reproductive toxicity study (ICI, 1988b, as cited by

ECHA, 2018; U.S. EPA, 1992). In reproductive toxicity studies, decreased body weight was

reported at 2000 mg/kg-day in rats gavaged for about 3 weeks, including after mating (Wato et

al., 2009), and at about 1600 mg/kg-day in rats exposed in the diet for 4 weeks (Nabae et al.,

2006; Kang et al., 2006).

OECD (2000) noted that hepatic hypertrophy and increased peroxisomal enzyme activity can

occur in rats and mice within a week of treatment with 12,000 ppm in feed (OECD 2000, citing

CMA, 1982a, 1986, 1995). In a 28-day gavage study, increased liver weight was seen at 1000

26

mg/kg-day. Although peroxisome proliferation can occur in humans, current scientific opinion is

that it does not proceed to increased liver weight or tumors in humans.

One study (Miyata et al., 2006) reported increased relative kidney weight and eosinophilic and

hyaline droplets in male rats treated with 1000 mg/kg-day by gavage for 28 days. Although the

finding of increased hyaline droplets suggests a connection with male rat-related alpha-2u-

globulin nephropathy, there was no specific staining for this protein. More importantly,

increased kidney weights were observed in males at 200 mg/kg-day in the absence of increased

hyaline droplets, and in female rats at 1000 mg/kg-day. However, kidney histopathology was not

reported in any other study, and the only other report of increased kidney weight was in female

rats gavaged with 1000 mg/kg-day for 2 or 4 weeks (Wato et al., 2009).

Reproductive effects were not seen in rats in a 1-generation study with doses up to 12,000 ppm

in the diet (about 1000 mg/kg-day). Similarly, there were no histopathological lesions in the

reproductive organs in a 2-year bioassay in rats and mice at doses up to 25,000 ppm (NTP,

1982). In females, ovarian follicle atresia and prolonged estrus cycle occurred at gavage doses of

1000 mg/kg-day (Wato et al., 2009; Miyata et al., 2006). Unlike DEHP, no DEHA-induced

reproductive effects have been observed in males.

DEHA is not a teratogen. Decreased litter weight and pup weight gain was seen in a 1-generaton

reproductive toxicity study in rats at about 1000 mg/kg-day (ICI, 1988b, as cited by ECHA,

2018; U.S. EPA, 1992; OECD, 2000). Other minor variations were seen in the offspring of rats

treated with the same dose of DEHA in the diet (ICI, 1988a, as cited by Versar, 2010; ECHA,

2018). Increased postnatal mortality was seen in the offspring of rats gavaged with 400 mg/kg-

day on GD 7 to PND 17 (Dalgaard et al., 2002, 2003). No adverse effects were seen in a

developmental study in rabbits at the highest tested dose of 145 mg/kg-day (Anonymous, 2014,

as cited by ECHA, 2018).

Based on the weight of evidence, DEHA is not mutagenic. It did not cause gene mutations in

bacterial or mammalian cells, or chromosome aberration in in vitro studies (Seed, 1982; Simmon

et al., 1977; Zeiger et al., 1985; McGregor et al., 1988; Galloway et al., 1987; Reisenbichler and

Eckl, 1993). DEHA was also marginal or negative for induction of chromosome aberrations and

micronuclei in vivo (Shelby et al., 1993; Shelby and Witt, 1995). Interpretation of the results of a

dominant lethal assay (Singh et al., 1975) is complicated by nonstandard reporting.

DEHA increased the incidence of hepatocellular adenomas and carcinomas in mice, but not rats.

These tumors have been shown to be due to a PPARα-related MOA (Lake et al., 1997), and so

are considered not relevant to humans (Felter et al., 2018).

5.10 Uncertainties and Data Gaps

The overall database on DEHA is extensive, including at least one of all key study types, and

numerous supplemental mechanistic and specialized studies. Subchronic and chronic bioassays

are available in rats and mice (NTP, 1982), although the extent of endpoint evaluation was less

thorough than modern standards, particularly for the subchronic studies. Two-generation

reproductive toxicity studies are not available, but a guideline-compliant unpublished 1-

27

generation reproductive study is available (ICI, 1988b, as cited by ECHA, 2018; U.S. EPA,

1992; OECD, 2000). This information is supplemented by specialized studies evaluating

reproductive effects in males (Nabae et al., 2006; Kang et al., 2006; Dalgaard et al. 2002, 2003),

females (Wato et al., 2009), or both (Miyata et al., 2006). In addition, standard guideline-

compliant developmental toxicity studies are available in rats (ICI, 1988a, as cited by Versar,

2010; ECHA, 2018) and rabbits (Anonymous, 2014, as cited by ECHA, 2018).

The only significant data gap was the absence of repeated dose data via the dermal and inhalation

routes.

Hazard:

Liver: The PPAR MOA is well-understood, and considered to lack human relevance. No

significant uncertainties were identified.

Kidney: There is some uncertainty associated with the kidney effects of DEHA, since

they were seen in only two studies (Miyata et al., 2006; Wato et al., 2009). There is also

uncertainty regarding a potential association of the kidney effects with male rat-related alpha-2u-

globulin nephropathy, although some similar effects were seen in female rats, or in the absence

of hyaline droplets (Miyata et al., 2006).

Developmental: There is some uncertainty regarding the toxicological significance of the

minor developmental variations noted in rats (ICI, 1988a, as cited by Versar, 2010; ECHA,

2018).

Cancer: The PPAR MOA is well-understood, and considered to lack human relevance.

No significant uncertainties were identified.

28

Table 3. Summary of NOAELs/LOAELs Identified for DEHA by Organ System

Species (Sex),

Reference

Exposure

Regimen

Effect

Category

Toxicological

Endpoint (mg/kg-

day)8

Toxicological Basis Comments

Fischer 344

(M&F)

50/sex/dose

NTP (1982)

103 weeks

Diet

0, 12,000,

25,000 ppm

(0, 948, 1975

mg/kg-day

(M); 0, 1104,

2300 mg/kg-

day (F)

Body weight NOAEL = 948 (M),

1104 (F)

LOAEL = 1975

(M), 2300 (F)

Reduced growth

throughout study

Decrease estimated from graph as

>10%

Liver NOAEL = 1975

M), 2300 (F)

LOAEL = N/A

No effects reported

Cancer NOAEL = 1975

M), 2300 (F)

LOAEL = N/A

Tumors that were noted were those

seen routinely in this strain of rat, and

they occurred in comparable numbers

in control and dosed rats.

B6C3F1

(M&F)

50/sex/dose

NTP (1982)

103 weeks

Diet

0, 12,000,

25,000 ppm

(0, 2040, 4250

mg/kg-day,

M&F)

Body weight NOAEL = 2040

(M), N/A (F)

LOAEL = 4250

(M), 2040 (F)

Decrease compared to

controls of >10%

Estimated from graph; no statistical

analysis or quantitative data

provided. Body weight in control

males varied substantially over time.

Liver NOAEL = 4250 (M

&F)

LOAEL = N/A

No changes in liver weight or

histopathology reported

Cancer Statistically

significant increase

at 4250 in males

and 2040 in

females; dose-

related increase in

males

Basis for cancer assessment (WOE

and OSF) on IRIS (U.S. EPA, 1991).

Increased incidence of liver tumors

(combined hepatocellular adenomas

and carcinomas) was observed in

8 All effect levels as identified by the authors of this assessment. Effect levels identified by previous assessments are in the comments column. N/A = not

applicable.

29

Species (Sex),

Reference

Exposure

Regimen

Effect

Category

Toxicological

Endpoint (mg/kg-

day)8

Toxicological Basis Comments

high dose males and all treated

females.

Reproductive/Developmental Toxicity

Wistar rat

(M&F)

15M+ 30F/

dose

OECD Guideline

415

ICI, 1988b, as

cited by ECHA,

2018; U.S. EPA,

1992; OECD,

2000

10 weeks

before mating

through

lactation

Diet

0, 300, 1800,

12,000 ppm

(0, 28, 170,

1080 mg/kg-

day)

Body weight NOAEL = 170 (F)

LOAEL = 1080 (F)

Decreased maternal body

weight gain during

gestation,

Co-principal study for the RfD on

IRIS (U.S. EPA, 1992)

Liver NOAEL = 170

(M&F)

LOAEL = 1080

(M&F)

Increased absolute and

relative weight,

considered associated with

peroxisome proliferation

Not relevant to humans

Developmental NOAEL = 170

LOAEL = 1080

Decreased litter weight,

and offspring weight gain

Decreased pup weight may have been

secondary to decreased maternal

weight gain, or may have been direct

toxic effect

Reproduction NOAEL = 1080

(M&F)

LOAEL = N/A

No effects on fertility,

reproductive organs,

Sprague-Dawley

rat

(M&F)

10/sex/dose

Miyata et al.,

2006

GLP Compliant,

28 days

Gavage in

corn oil

0, 40, 200,

1000 mg/kg-

day

Body weight NOAEL (M, F) =

1000

LOAEL = N/A

Study designed to investigate effects

on hormones and reproductive organs

in males and females.

Liver weight change potentially

adverse in light of peroxisome

proliferative potential

Liver NOAEL = 200

LOAEL = 1000

Increased relative liver (M

and F)

Kidney NOAEL = 40 (M)

LOAEL = 200 (M)

NOAEL = 200 (F)

LOAEL = 1000 (F)

Increased relative kidney

weight. Also, increased

eosinophilic and hyaline

droplets

30

Species (Sex),

Reference

Exposure

Regimen

Effect

Category

Toxicological

Endpoint (mg/kg-

day)8

Toxicological Basis Comments

OECD Guideline

407 compliant

Adrenal NOAEL = 200 (F)

LOAEL = 1000 (F)

NOAEL = 1000

(M)

LOAEL = N/A (M)

Increased relative adrenal

weight

Kidney findings are suggestive of

male rat-related alpha-2u-globulin

nephropathy, there was no specific

staining for this protein, and

increased kidney weight was seen in

females and in the absence of hyaline

droplets.

Reproductive

(M)

NOAEL = 200 (F)

LOAEL = 1000 (F)

NOAEL = 1000

(M)

LOAEL= N/A (M)

No effects in males

Ovarian follicle atresia

and prolonged estrus cycle

in females

Sprague-Dawley

rat

(F)

10/dose

Wato et al., 2009

2 or 4 weeks

Gavage

0, 200, 1000

or 2000

mg/kg-day

Liver NOEL = 200

Increased relative liver

weight at 2 and 4 weeks

No evaluation of histopathology, but

increase at 1000 was only 12%

Kidney NOAEL = 200

LOAEL = 1000

Increased relative kidney

weight at 2 and 4 weeks

Eosinophilic change of proximal

tubule were observed at 2000 mg/kg-

day in the 2-week study and 1000

mg/kg-day and above in the 4-week

study

Reproductive NOAEL = 200

LOAEL = 1000

Decrease in relative ovary

weight, increased large

follicle atresia

Mean estrous cycle length was

reduced at 200 mg/kg-day, but not at

higher doses

Sprague-Dawley

(Crj:CD) rat

(F)

2 weeks

before mating

through GD 7

Body weight NOAEL = 1000

LOAEL = 2000

Decreased body weight

and body weight gain

prior to the mating period,

but not during gestation

No effect on food consumption

31

Species (Sex),

Reference

Exposure

Regimen

Effect

Category

Toxicological

Endpoint (mg/kg-

day)8

Toxicological Basis Comments

10 F/dose mated

to untreated

males

Wato et al, 2009

Gavage in

Corn Oil

0, 200, 1000,

2000 mg/kg-

day

Reproductive NOAEL = 200

LOAEL = 1000

Follicle atresia in the

ovary; significant increase

in mean estrus cycle

length; decreased corpus

luteum formation and

increased follicular cyst

Development NOAEL = 200

LOAEL = 1000

Increased pre- and post-

implantation loss,

decreased number of live

embryos

Fisher 344 Rat

(M)

6/dose

Nabae et al.,

2006

4 weeks

Diet

0, 6000,

25,000 ppm

(0, 318, 1570

mg/kg-day)

Body weight NOAEL = 318

LOAEL = 1570

Study designed to investigate

testicular effects

Relative testes weight was increased,

but not considered adverse because

relative testes weight was increased

rather than decreased (possibly

secondary to reduced body weight)

and not accompanied by abnormal

spermatogenesis or testicular

histopathology findings.

Reproductive NOAEL = 1570

LOAEL = N/A

Fisher 344 Rat

(M),

6/dose

Kang et al., 2006

4 weeks

Diet

0, 6000,

25,000 ppm

Body weight NOAEL = 318

LOAEL = 1570

Study designed to investigate

testicular effects

Relative liver weight increased by

50% at high dose

Reproductive NOAEL = 1570

LOAEL = N/A

32

Species (Sex),

Reference

Exposure

Regimen

Effect

Category

Toxicological

Endpoint (mg/kg-

day)8

Toxicological Basis Comments

(0, 318, 1570

mg/kg-day)

No effect on testes weight

Wistar rat

8 dams/dose

Dalgaard et al.

2002, 2003

GD 7 to PND

17

0, 800, or

1200 mg/kg-

day

Gavage in

peanut oil

Maternal NOAEL = 800

LOAEL = 1200

Decreased maternal body

weight gain during GD 7-

21, increased pregnancy

length

Developmental NOAEL = N/A

LOAEL = 800

Body weight of M and F

pups decreased on PND 3

(the only PND evaluated)

Increased percentage of perinatal loss

at 1200 mg/kg-day. Body weight

also decreased at birth at 1200

mg/kg-day.

No anti-androgenic effects

Wistar Rat

20 dams/dose

Dalgaard et al.,

2002, 2003

GD 7 to PND

17

Gavage in

peanut oil

0, 200, 400,

800, 1200

mg/kg-day

1/sex/litter

retained until

adulthood for

measurement

of sexual

maturation

Maternal NOAEL = 400

LOAEL = 800

Increased gestation length Study designed to investigate anti-

androgenic and other developmental

effects of perinatal exposure; no anti-

androgenic effects observed Developmental NOAEL = 200

LOAEL = 400

Increased postnatal

mortality

Wistar Rat (F)

24 dams/dose

GD 1-22

Maternal

effects

NOAEL = 170

LOAEL = 1080

Decreased food

consumption and body

33

Species (Sex),

Reference

Exposure

Regimen

Effect

Category

Toxicological

Endpoint (mg/kg-

day)8

Toxicological Basis Comments

ICI, 1988a, as

cited by Versar,

2010; ECHA,

2018

OECD Guideline

414 and GLP

compliant

Diet

0, 300, 1800

or 12000 ppm

Reported by

authors as 0,

28, 170, 1080

mg/kg-day

weight gain (-13%)

throughout gestation

Standard teratogenicity study. Co-

principal study for the RfD on IRIS

(U.S. EPA, 1992).

The study authors considered the low

dose to be a developmental NOAEL,

while EPA considered the mid dose a

NOAEL, and ECHA considered the

high dose a NOAEL. It is not clear

how the incidence of the variations

differed at the mid and high doses.

Fetal

development

NOAEL = 28-170

LOAEL = 1080

Minor skeletal variations

(delayed ossification) and

visceral variations (kinked

ureter and slightly dilated

ureter)

New Zealand

White Rabbit (F)

21 to 27/dose

Anonymous

(2014), as cited

by ECHA (2018)

OECD Guideline

414 and GLP

compliant

Days 6 to 29

post-coitum

Diet

Concentration

in diet not

reported;

Target doses

of 0, 40, 80

and 160

mg/kg-day;

actual intake

was 0, 36, 70,

and 145

mg/kg-day

Maternal NOAEL = 145

LOAEL = N/A

No adverse effects. ECHA (2018), concluded there were

no adverse effects based on clinical

observations, body weight changes,

food/water consumption, mortality

and effects on ovaries and uterus.

ECHA (2018) concluded there were

no toxicologically relevant effects on

litter size, sex ratio, fetal body

weight, external, visceral, or skeletal

malformations or variations

Developmental NOAEL = 145

LOAEL = N/A

No adverse effects

34

6 Exposure

The use of DEHA in consumer products has been described in Section 3 of this report.

There are limited data on DEHA residues in products or in environmental compartments. DEHA

was found in 1 of 41 children’s products in a CPSC 2002 evaluation of children’s soft PVC

articles (Chen, 2002), but not in a more recent study (Dreyfus, 2010, as cited by CPSC, 2014). In

Japan, 8.5% of products that children often mouth or hold contained DEHA (Kawakami et al.,

2011, as cited by Bui et al., 2016). Bui et al. (2016) also reported that DEHA was found in new

infant crib mattress covers (4.8 mg/g material, 11.1% detection frequency) and the breathing

zones of sleeping infants contained an average concentration of 8.4 μg/m3 (Boor et al., 2015;

Liang and Xu, 2014; as cited by Bui et al., 2016). Liang and Xu (2014, as cited by Bui et al.,

2016) estimated emissions of DEHA from crib mattress covers at different temperatures, and

used the resulting data to validate an emission model for predicting concentrations in indoor air.

Using the model, they estimated a concentration of 1.05 μg/m3 in indoor air (presumably the

entire well-mixed room).

Remberger et al. (2005) reported that no adipates, including DEHA, were detected in air or

human breast milk (Remberger et al. 2005). DEHA metabolites were not investigated in that

study. However, a more recent study found DEHA in breast milk at 2 µg/L (additional methods

information not available in the secondary source) (Palm-Cousins et al., 2007, as cited by Bui et

al., 2016). Other measurements reported DEHA in dust (2–10 μg/g) and indoor air (5–15 ng/m3)

(Rudel et al., 2003, as cited by Bui et al., 2016).

DEHA is used as a plasticizer in various food storage wraps and it has been shown to migrate

into stored foods; thus the general population can be exposed through consumption of foods

stored in plastic films (HSDB, 2008), and this can be a major source of general population

exposure. For example, in a migration study by Petersen and Naamansen (1998), DEHA

migration into fresh meat from food packaging was measured between 1 and 40 mg/kg

depending on fat content and number of times the meat was sliced and repacked in the DEH-

containing film. Higher temperatures and microwave cooking of foods can also enhance

migration (Startin et al., 1982, as cited by OECD, 2000).

Data on exposure to DEHA from toys and child care articles in the U.S. are not available (CPSC,

2014). Bui et al. (2016) summarized several studies of total DEHA intake, however. Estimates

included 1 µg/kg-day for children aged 15–20 months old, from the diet (Fromme et al., 2013),

infant intake of 2.35 µg/kg-day from inhalation (Liang and Xiu, 2014), adult dietary intake of

0.67 µg/kg-day (Fromme et al., 2007), and 0.46 µg/kg-day from oral, inhalation and dermal

exposure of an unspecified population (Stuer-Lauridsen et al., 2001).

Fromme et al. (2007) also quantified the median dietary intake of DEHA in a European

population as 0.7 µg/kg-day (27 female and 23 male subjects aged 14-60 years). The median, as

well as the 95th percentile daily dietary intake, did not exceed the recommended tolerable daily

intake (Fromme et al., 2007). In one-week duplicate diet samples provided by three Japanese

hospitals, Tsumura et al. (2003) determined a total mean daily intake of DEHA as 12.5 µg.

35

Dietary exposures have also been estimated for Canadian populations as 137 to 259 μg/kg-day

(Page and Lacroix, 1995; Carlson and Patton, 2012).

Inhalation of indoor air in office buildings using DEHA-containing plastics is another route of

human exposure (HSDB, 2008). Based upon indoor air monitoring of an office building, the

representative indoor air concentration of DEHA was determined to be 2.0 ng/m3; the source of

the DEHA exposure was thought to be from plasticizer use (HSDB, 2008).

Widespread use of DEHA has made its investigation alongside phthalates in exposure and

leaching studies commonplace (Cao, 2008; Fromme et al., 2007; Kueseng et al., 2007; Tsumura

et al., 2003). Additionally, heavy and widespread use in food packaging and other industries has

led to widespread human exposure to this chemical (Remberger et al., 2005). In this study,

conducted by the Swedish Environmental Research Institute, eight adipates were screened for in

air, water, sediment, sludge, biota and human breast milk. DEHA was the only adipate frequently

detected in samples. That is, it was detected in the majority of the samples, compared to the

seven other adipates tested, five of which were not detected at all. Two were detected in sludge.

Only limited information was located on concentrations of DEHA in environmental media.

OECD (2000) summarized environmental monitoring data collected by Felder et al. (1986) and

reported by Hicks and Michael (1983). The study evaluated water and sediment samples

collected at 24 sites in the US. Of the 85 water samples, 6 exceeded the detection limit of 0.2

μg/L, with a maximum reported value of 1.0 μg/L from one of four replicates at one site (and the

other three replicates not exceeding the detection limit). The geometric mean concentration of

DEHA in sediments was <0.8 mg/kg dry weight. Air emission in 1994 in the U.S. was estimated

as 315,000 kg (U.S. EPA, 1996, as cited by Bui et al., 2016).

Occupational exposure to DEHA occurs during its production, its use as a plasticizer, and its use

as a lubricant and functional fluid (IARC, 1982). Exposure can occur through dermal contact and

inhalation (IARC, 1982). OECD (2000, source not provided) reported that, based on an uncited

survey of manufacturers, it is estimated that only 25-50 individuals in the US are involved in the

manufacturing and handling process for DEHA. However, occupational exposure also includes

users of DEHA-containing products, and so is a much larger population. The NIOSH NOES

Survey (NIOSH, 1983) has statistically estimated that 15,636 workers (3,628 of these are female)

are potentially exposed to DEHA in the U.S. For example, the average concentration of DEHA

in the air of a meat-wrapping department of a supermarket, as a result of heating polyvinyl

chloride film during meat packaging operations, was estimated to be 0.014 ppm (0.2 mg/m3)

(IARC, 1982).

The U.S. EPA has set a regulatory threshold for DEHA in water and toxicological threshold for

risk purposes. The U.S. EPA Maximum Contaminant Level (MCL) for DEHA in drinking water

is 0.4 mg/L (U.S. EPA, 2012), and the oral reference dose (RfD) is 0.6 mg/kg-day (U.S. EPA,

1992).

Biomonitoring

No biomonitoring data was identified for DEHA.

36

7 Discussion

7.1 Toxicity Under FHSA

Animal data were sufficient to support the conclusion that DEHA does not fit the designation

of “acutely toxic” under the Federal Hazardous Substances Act (FHSA) (16

CFR§1500.3(c)(2)(i)(A)) following single oral or dermal exposures. The oral LD50 is >20,000

mg/kg in rats (NTP, 1982). The dermal LD50 of DEHA in rabbits is >8000 mg/kg (Smyth et al.,

1951; OECD, 2005, as cited by NICNAS, 2011), but the available studies were not well-

documented. No inhalation LC50 is available, but no mortality occurred in rats exposed to 5.7

mg/L DEHA aerosol for 4 hours (1998 study cited by ECHA, 2018). DEHA is minimally

irritating to skin and eyes (CTFA, 1967; Anonymous, 1984), and not a sensitizer (ECHA, 2018).

Dermal phototoxicity tests of DEHA in humans and rabbits showed no phototoxic (primary

irritant) or photoallergic reactions (Anonymous, 1984 as cited in Versar, 2010).

The systemic toxicity of DEHA following repeated dosing is low. The major effects observed are

decreased body weight and increased liver weight related to peroxisome proliferation. There are

also sporadic reports of increased kidney weight.

Anti-androgenic effects have not been seen with DEHA. Reproductive effects were seen only in

females and were limited to ovarian follicle atresia and prolonged estrus cycle (Wato et al., 2009;

Miyata et al., 2006).

DEHA is not a teratogen. Decreased litter weight and pup weight gain was seen in a 1-generaton

reproductive toxicity study in rats (ICI, 1988b, as cited by ECHA, 2018; U.S. EPA, 1992;

OECD, 2000), and minor variations were seen in the offspring of treated rats (ICI, 1988a, as

cited by Versar, 2010; ECHA, 2018). There was also a report of increased postnatal mortality

was seen in the offspring of rats gavaged on GD 7 to PND 17 (Dalgaard et al., 2002, 2003).

Based on the weight of in vitro and in vivo evidence, DEHA is not mutagenic.

DEHA is a mouse hepatocarcinogen via a PPAR MOA that is well understood (Lake et al., 1997)

and not considered relevant to humans (Felter et al., 2018).

37

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47

APPENDIX 1

Search Terms Used

“Bis(2-ethylhexyl) hexanedioate” OR “Di-(2-ethylhexyl) adipate” OR “Dioctyl adipate” OR

“Hexanedioic acid, bis(2-ethylhexyl) ester” OR “Adipic acid, bis(2-ethylhexyl) ester” OR

“Bis(2-ethylhexyl) adipate” OR “DEHA” OR "Di(2-ethylhexyl) adipate" OR "Di(2-

ethylhexyl)adipate" OR "Di-2-ethylhexyl adipate" OR “Hexanedioic acid, dioctyl ester” OR

"Hexanedioic acid, 1,6-bis(2-ethylhexyl) ester" OR “Octyl adipate” OR (103-23-1)

48

APPENDIX 2

Explanation of Physico-chemical Parameters

The organic carbon normalized solid-water partition coefficient (Koc), also known as the organic

carbon adsorption coefficient, is defined as the ratio of the chemical’s concentration in a state of

sorption (i.e. adhered to soil particles) and the solution phase (i.e. dissolved in the soil water).

Koc is crucial for estimating a chemical compound's mobility in soil and the prevalence of its

leaching from soil. For a given amount of chemical, the smaller the Koc value, the greater the

concentration of the chemical in solution. Thus, chemicals with a small Koc value are more likely

to leach into groundwater than those with a large Koc value

(http://www.acdlabs.com/products/phys_chem_lab/logd/koc.html ).

Henry's law, one of the gas laws formulated by William Henry, states that “at a constant

temperature, the amount of a given gas dissolved in a given type and volume of liquid is directly

proportional to the partial pressure of that gas in equilibrium with that liquid

(http://en.wikipedia.org/wiki/Henry's_law).” Henry's Law Constants characterize the equilibrium

distribution of dilute concentrations of volatile, soluble chemicals between gas and liquid phases

(http://www.epa.gov/athens/learn2model/part-two/onsite/esthenry.htm).

The octanol/water partition coefficient (Kow) is defined as the ratio of a chemical's concentration

in the octanol phase to its concentration in the aqueous phase of a two-phase octanol/water

system. In recent years, this coefficient has become a key parameter in studies of the

environmental fate of organic chemicals. It has been found to be related to water solubility,

soil/sediment adsorption coefficients, and bioconcentration factors for aquatic life. Because of its

increasing use in the estimation of these other properties, Kow is considered a required property

in studies of new or problematic chemicals

(http://www.pirika.com/chem/TCPEE/LOGKOW/ourlogKow.htm).

The bioconcentration factor (BCF) is the concentration of a particular chemical in a tissue per

concentration of chemical in water (reported as L/kg). This property characterizes the

accumulation of pollutants through chemical partitioning from the aqueous phase into an organic

phase, such as the gill of a fish. The scale used to determine if a BCF value is high, moderate or

low will depend on the organism under investigation. The U.S. EPA generally defines a high

potential BCF as being greater than 5,000; a BCF of moderate potential as between 5,000 and

100; a low potential BCF as less than 100 (http://en.wikipedia.org/wiki/Bioconcentration_factor;

http://sitem.herts.ac.uk/aeru/footprint/en/Quest/ecotox.htm).